Since its discovery, the only known mechanism of signaling for cAMP involves its binding to the regulatory (R) subunit of the cAMP-dependent protein kinase (PKA) that leads to the dissociation of the holoenzyme and activation of the catalytic (C) subunit kinase. There have been speculations that the R subunit of PKA may have other functions in addition to inhibiting the C subunit kinase activity. However, evidence linking a function to the R subunit has been elusive.
Signal Transduction Pathway of cAMP
The cAMP-signal-transduction-pathway-mediated phosphorylation can be elicited by various physiological ligands in cells and is critically involved in the regulation of metabolisms, cell growth and differentiation, apoptosis, and gene expression. The PKA holoenzyme is composed of two genetically distinct subunits, catalytic (C) and regulatory (R), forming a tetrameric holoenzyme R2C2 which, in the presence of cAMP, dissociates into an R2(cAMP)4 dimer and two free catalytically active C subunits. There are two major R subunit isoforms which are further distinguished as RIα and RIβ, and RIIα and RIIβ, and three isoforms of the C subunit, Cα, Cβ, and Cγ. Defects in the formation or action of cAMP may cause cellular transformation. (Cho-Chung, Y. S. (1990) Cancer Res., 50:7093–7100; Gottesman, M. M., and Fleischmann, R. D. (1986) Cancer Surveys, 5:291–308). Furthermore, differential expression of RI and RII has been correlated with cell differentiation and neoplastic transformation. In fact, while RI is preferentially expressed in transformed cells, expression of RII is increased in terminally differentiated tissues. (Cho-Chung, Y. S. (1990) Cancer Res. 50:7093–7100).
Mechanisms of cAMP Signaling
For approximately forty years, the R subunit has been the only known receptor for cAMP in cells and cAMP binding to the holoenzyme has been the accepted mechanism that regulates PKA activity. However, this dogma of cAMP signaling is being rewritten to accommodate some recent discoveries that implicate the existence of alternative mechanisms for the cAMP messenger system (FIG. 1). The first hint of a novel alternative mechanism for cAMP signaling came from studies that show the direct interaction of cAMP with some ion channels in the central nervous system (Liu, F. C., et al (1995) J. Neurosci, 15:2367–2384; Zufall, F. et al, (1997) Curr. Opin. Neurobiol., 7:404–412; Santoro, B. et al (1998) Cell, 93:717–729)., suggesting that there are receptors, other than the R subunit, that mediate the action of cAMP. This was followed by a study that demonstrated that the C subunit can be activated in a cAMP- and R subunit-independent manner, in a ternary complex of NFκB-IκB-C subunit (Zhong, H. et al (1997) Cell 89:413–424). Degradation of IκB following the exposure to inducers of NFκB leads to the activation of the C subunit in a cAMP-independent manner and subsequent phosphorylation of NFκB. Recently, a novel family of cAMP-binding guanine nucleotide exchange factors was identified which can selectively activate the Ras superfamily of guanine nucleotide binding protein Rap 1 in a cAMP-dependent but PKA independent manner (De Rooij, J. et al (1998) Nature 396:474–477; Kawasaki, H. et al (1998) Science 282:2275–2279).
Functions for the Regulatory Subunit
There has also been speculation that the R subunit could act through mechanisms other than C subunit activation. One possibility is that R subunit containing bound cAMP has functions independent of its interaction with the C subunit. For example, cAMP-bound RII subunit complex but not the C subunit nor the protein kinase holoenzyme inhibits phosphorylase phosphatase activity, leading to prolongation of the glycogen breakdown cascade (Gergley, P. and Bot, G. (1997) FEBS Letters 82:269–272). Gergley et al. suggested that the inhibition of phosphorylase phosphatase activity by the R subunit was through a substrate-directed mechanism perhaps through conformational modification of phosphorylase a. The RII subunit also inhibits the activity of a purified high molecular weight phosphoprotein phosphatase in a cAMP-dependent manner and that the inhibited species is an RII-cAMP-phosphatase complex (Khatra, B. S. et al (1985) Biophy. Res. Comm. 130:567–572). By inhibiting phosphatase activity, the R subunit may magnify the effect of C subunit phosphorylation. In addition, the RII subunit associates with numerous binding proteins known as the A-kinase anchoring proteins (AKAPs), which serve to localize the inactive PKA holoenzyme in specific subcellular compartments (Dell'Acqua, M. L. and Scott, J. D. (1997) J. Biol. Chem. 272:12881–12884; Pawson, T. and Scott, J. D. (1997) Science 278:2075–2080). These studies together suggest that the R subunit may interact with other proteins in addition to the C subunit.
Recently, it was also found that RIα interacts with the ligand-activated epidermal growth factor receptor (EGFR) complex (Tortora, G. et al (1997) Oncogene 14:923–928). Coimmunoprecipitation with an anti-RIα antibody demonstrated the binding of RIα to the SH3 domains of the Grb2 adaptor protein, allowing the localization of the type I PKA to the activated EGFR (Tortora, G. et al (1997) Oncogene 14:923–928). Using affinity chromatography and immunoprecipitation, another study provided evidence for a direct interaction between RIα and the p34cdc2 protein kinase cell cycle regulator, presenting the possibility of interdependent functioning of these two pathways in the regulation of cell division (Toumier, S. et al (1991) J. Biol. Chem. 266:19018–19022).
The role of cAMP in cell growth has been widely studied (Cho-Chung, Y. S. (1990) Cancer Res. 50:7093–7100). In a large number of human cancer cell lines, RI isoform is the only R subunit of PKA detected. In human cancer specimens, the predominant expression of type I PKA or the RI subunit is consistently observed. It has been shown that overexpression of RIα in Chinese hamster ovary (CHO) cells rendered growth advantages in monolayer and soft agar conditions, whereas overexpression of the C subunit did not produce such consequences (Tortora, G. et al (1994) Int. J. Cancer 59:712–716). Similarly, overexpression of RIα, but not the C subunit, in MCF-10A cells conferred the ability to grow in serum and growth-factor free conditions (Tortora, G. et al (1994) Oncogene 9:3233–3240). It is apparent from these studies that the role of cAMP in cell growth cannot be explained by changes in the kinase activity and further raises the possibility that the R subunit or an unidentified cAMP receptor molecule may mediate the effects of cAMP.
Cyclic AMP Signaling and Gene Regulation
In eukaryotes, transcriptional regulation by the cAMP signaling pathway is mediated by a family of cAMP-responsive nuclear transcription factors (Lalli et al. (1994) J. Biol. Chem. 269:17359–17362; Daniel et al. (1998) Aannu. Rev. Nutr. 18:353–383). These factors may act as either activators or repressors and they contain signature basic domain/leucine zipper motifs and bind as dimers to cAMP-responsive elements (CRE). The consensus CRE has the nucleotide sequence TGACGTCA as found in many promoters of cAMP regulated genes. The CRE-binding proteins (CREB) and modulators (CREM) are regulated by phosphorylation by PKA. Binding of cAMP to the R subunits releases the C subunits, thus enabling a fraction of the C subunit to enter the nucleus and phosphorylate its target proteins which include a large number of the CREB and CREM family of proteins. CREB and CREM belong to a group of transcription factors that contain basic region leucine zippers, bZIP, which is central for DNA recognition and binding, and protein-protein interaction (homo- and heterodimerization) among family members. In addition, a kinase-inducible domain (KID) also known as the phosphorylation box (P-box), contains potential phosphorylation sites for PKA and several different kinases that are critical for the transactivation properties of CREB and CREM. The phosphorylation of CREB/CREM within the KID domain induces their association with transcriptional coactivators, such as the nuclear factor CBP (CREB binding protein) or its closely related but distinct nuclear factor p300.
Several other transcription factors are also regulated by and are responsive to the activation of the cAMP signaling pathway, including the activating transcription factor-1 (ATF-1), NFκB, AP-2, and some nuclear receptors (Daniel et al. Supra.). Of specific interest is NFκB, which is a cytoplasmically localized transcription factor and may be directly controlled by cAMP (Naumann et al. (1994) EMBO J 13:4597–4607; Neumann (1995) EMBO J 14:1991–2004). Elevation of cAMP levels can either activate or inhibit NFκB regulated gene expression. Furthermore, there is also evidence that signals that cause degradation of IκB allows the complexed C subunit to phosphorylate NFκB and further activates NFκB and its translocation into the nucleus (Zhong et al. (1997) Cell 89:413–424). As alluded to above, the RIIβ subunit can also act directly as a transcription activator of CRE-regulated gene expression.
PKA Signaling in Yeast
In the yeast S. cerevisiae, PKA activity has been implicated in numerous cellular processes, including growth, carbon storage, response to stress and differentiation (Cameron et al. (1988) Cell 53:555–566; Broach et al (1990) Adv. Cancer Res. 54:79–138; Gimeno et al. (1992) Cell 68:1077–1090). In contrast to mammalian cells, the R subunit of PKA in yeast is encoded by the single BCY1 gene and the C subunits are encoded by three TPK genes (termed TPK1, TPK2, and TPK3) (Matsumato et al (1985) Yeast 1:15–24; Cannon et al. (1987) Mol. Cell Biol. 7:2653–2663; Toda et al (1987) Mol. Cell Biol 7:1371–1377; Toda et al. (1987) Cell 50:277–287). In S. cerevisiae, exposure to mild stress leads to development of tolerance against higher doses of the same stress and also cross tolerance to stress caused by other agents. Stress initiates expression of genes encoding proteins with stress-protective functions. Transcriptional control by multiple stress conditions is mediated by the stress response element (STRE) (Moskovina et al. (1999) Mol. Microbiol. 32:1263–1272). S. Cerevisiae PKA acts as a powerful repressor of STRE-mediated transcription (Moskovina, Supra.; Smith et al. (1998) EMBO J. 17:3556–3564). It appears to provide a link between positive control of cell growth and negative control of stress response.
Although the precise mechanism of the general stress response pathway has not been elucidated, recent studies have implicated the related zinc finger transcription factors Msn2p and Msn4p in this process (40–42). Strains lacking MSN2 and MSN4 are sensitive to various forms of stress and fail to accumulate stress-regulated messages following heat and osmotic stress, as well as nutrient starvation and DNA damage. Furthermore, it has been shown that Msn2p and Msn4p can recognize and bind STREs in vitro (40,41). These proteins appear to be functionally redundant, as double but not single mutants exhibit pleiotropic stress sensitivity. Msn2p seems to have a more pronounced role, but full stress-induced expression of STRE-regulated genes is dependent on the presence of both Msn2p and Msn4p. Msn2p/Msn4p relocate from the cytoplasm and accumulate in the nucleus under stress conditions. Nuclear localization of Msn2p/Msn4p is inversely correlated with cAMP levels and PKA activity (43). It is intriguing that the response to multiple stresses and to PKA activity can be mediated by only one type of transcription factor. In mammalian cells, pathways linking transcriptional response to multistress mediated by factors shutting between cytoplasm and nucleus, have not been explored. The presence of a comparable cAMP-regulated multistress response pathway and the Msn2p/Msn4p shuttling factors in higher eukaryotes remains an exciting possibility.
Cis Platin Resistance and Regulation of DNA Repair in cAMP-Dependent Protein Kinase Mutants
It has been demonstrated that the mouse Y1 adrenocortical carcinoma and CHO cells harboring defective RIα subunits of PKA, with decreased kinase activity, exhibit increased resistance to cisplatin (Liu, B. et al (1996) Cell growth and Differ. 7:1105–1112). In contrast, C subunit mutants also with diminished response to cAMP and decreased kinase activity, have similar sensitivity to cisplatin as wild-type cells, suggesting that the R subunit may confer resistance independent of the C subunit kinase activity. Moreover, wild-type cells transfected with a mouse dominant mutant RIα cDNA are also more resistant to cisplatin than wild-type cells. In addition, increased nuclear protein binding to cisplatin-damaged DNA was observed with nuclear extracts from RIα mutant compared to wild-type and C subunit mutants. A host cell reactivation assay also indicate that RIα mutant repairs and reactivates a cisplatin damaged reporter plasmid more efficiently than wild-type cells and the C subunit mutant. These results suggest that alteration specifically in the RIα subunit, but not the C subunit nor the kinase activity, confers cellular resistance to cisplatin. We further speculate that the RIα subunit may have other functions and regulate drug resistance.
Regulation of P-Glycoprotein Expression in cAMP-Dependent Protein Kinase Mutants
Additional evidence supporting a function for R subunit in drug resistanceis stemmed from studies on multidrug resistance (Cvijic, M. E. and Chin, K. V. (1997) Cell growth and Diff. 8:1243–1247). It has been shown that the RIα subunit mutants of CHO cells exhibited increased sensitivity to chemotherapeutic agents that are substrates for the multidrug transporter or P-glycoprotein (Abraham, I. et al (1987) Mol. Cell. Bio. 7:3098–3106; Abraham, I. et al (1990) Exp. Cell. Res. 189:133–141; Chin, K. V. et al (1992) J. Cell. Physiol. 152:87–94). The alteration in drug sensitivity in the RIα mutants resulted from a reduced expression of the multidrug resistance (mdr) gene. In the current study, we further examined the drug sensitivity and iP-glycoprotein levels in a series of C subunit mutants of the CHO cells. Our results revealed that these mutants exhibit similar sensitivity as wild-type cells to adriamycin, taxol and colchicine. Furthermore, no changes in P-glycoprotein expression was observed with these C subunit mutants compared to the wild-type cells. These results suggest that the decreased mdr gene expression in the RIα subunit mutants may be a result of the mutation and altered function of the RIα gene rather than alteration of the kinase activity, further supporting that RIα may regulate drug resistance independent of the kinase.
Effects of RIα Overexpression on Cisplatin Sensitivity in Carcinoma Cells
RIα has been overexpressed in the human ovarian carcinoma A2780 cells to demonstrate that modulating RIα levels can influence cellular sensitivity to cisplatin (Cvijic, M. E. and Chin, K. V. (1998) BBRC 249–723–727). Retroviralinfected A2780 cells overexpressing wild-type RIα cDNA displayed a 4- to 8-fold greater sensitivity to cisplatin as compared with parental cells. Overexpression of RIα in the CP70 cisplatin-resistant derivative of A2780 also increased the sensitivity of these cells to cisplatin. Therefore, enhanced expression of the RIα subunit of PKA sensitizes cells to the cytotoxic effects of this DNA-damaging agent. These data suggest that RIα may act directly, independent of the C subunit, to influence cellular sensitivity to cisplatin. Therefore, modulation of RIα expression or its functional status by pharmacological agents may be clinically useful in reverse cisplatin resistance in cancer.
Cisplatin Sensitivity of PKA Mutants in S. Cerevisiae 
The role of PKA in cellular sensitivity to cisplatin was evaluated in a series of PKA mutants of Saccharomyces cerevisiae (Cvijic, M. E. et al (1998) Anticancer Res. 18:3187–3192). Mutants with decreased kinase activity resulting from a srv2 mutation showed no alterations in cisplatin sensitivity. Complementation of TPK1, the yeast C subunit of PKA, in a mutant strain containing tpk1 and also tpk2 and tpk3 deletions did not significantly alter its sensitivity to cisplatin. Yeast transformants containing increased kinase activity resulting from overexpression of RAS2Val19 or TPK1 and yeast strains having increased kinase activities due to mutations in the R subunit, BCY1, gene also did not show alterations in their sensitivity to cisplatin. Therefore, these results unambiguously demonstrate that changes in PKA activity owing to either mutations in the C subunit or indirectly through alterations in other molecules of the cAMP signaling pathway, have no effect on cisplatin sensitivity in S. cerevisiae. 
PKA R Subunit Interacts with Cytochrome Oxidase Subunit Vb
To gain further understanding of the function of RIα, Yang et al performed the yeast two-hybrid interaction cloning experiments and showed that the RIα subunit associates with the cytochrome c oxidase subunit Vb (CoxVb) (Yang, W. L. et al (1998) Biochemistry 37:14175–14180). The mammalian cytochrome c oxidase, composed of 13 polypeptide subunits, is the terminal enzyme complex of the electron transfer chain that oxidizes cytochrome c and transfers electrons to molecular oxygen to form water and the synthesis of ATP. We show further that CoxVb interacts with the GST-RIα fusion protein and also coimmunoprecipitates RIα in cell extracts. Binding of CoxVb to RIα can be dissociated with cAMP. Treatment with cAMP-elevating agents inhibits cytochrome c oxidase activity in CHO cells with a concomitant decrease in cytochrome c levels in the mitochondria and an increase in its release into the cytosol. Furthermore, mutant cells harboring a defective RIα show increased cytochrome c oxidase activity and also constitutively lower levels of cytochrome c in comparison to either the wild-type cells or the C subunit mutant. These results suggest a novel mechanism of cAMP signaling through the interaction of RIα with CoxVb thereby regulating cytochrome c oxidase activity as well as the release of cytochrome.